The development of new biological materials, particularly those capable of serving as permissive substrates for cell growth, differentiation, and biological function, is a key area for advancing medical technology. Recently, attempts have been made to develop new biologically compatible scaffolds for controlled drug release, tissue repair, and tissue engineering. Since many diseases cannot be treated solely by small molecule drugs, researchers have begun investigating the potential role of biomaterials alone or in combination with cell-based therapies as an alternative therapeutic strategy.
Poloxamer 407 (pluronic F-127, PF-127) is a thermoreversible scaffold composed of polyoxyethylene-polyoxypropylene copolymers in a concentration ranging from 20-30%. (Miyazaki S, et al., “Pluronic F-127 gels as a vehicle for topical administration of anticancer agents,” Chem Pharm Bull (Tokyo) 1984, 32(10):4205-4208.) The amphiphilic nature of poloxamer 407 can allow its use as a drug carrier in a variety of settings including administration by oral, topical, intranasal, vaginal, rectal, ocular, and parenteral routes. (Escobar-Chavez J J, et al., “Applications of thermo-reversible pluronic F-127 gels in pharmaceutical formulations,” J Pharm Pharm Sci 2006, 9(3): 339-358.) The potential use of poloxamer 407 as an artificial skin has been reported, and there have been several studies on use of poloxamer 407 for in vivo tissue engineering of cartilage and lung. (DiBiase Md., Rhodes Conn., “Investigations of epidermal growth factor in semisolid formulations,” Pharm Acta Helv 1991, 66(5-6):165-169; Liu Y et al., “Repairing large porcine full-thickness defects of articular cartilage using autologous chondrocyte-engineered cartilage,” Tissue Eng, 2002, 8(4):709-721; Cortiella J et al., “Tissue-engineered lung: an in vivo and in vitro comparison of polyglycolic acid and pluronic F-127 hydrogel/somatic lung progenitor cell constructs to support tissue growth,” Tissue Eng, 2006 May, 12(5):1213-1225.)
It appears that poloxamer 407 not only facilitates tissue formation but also can be important for proper tissue assembly. (Cortiella et al. (2006).) Poloxamer 407 has also been reported to provide a 3D environment for differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into adipocytes, providing a potential alternative cell source for adipose tissue engineering. (Vashi A V et al., “Adipose differentiation of bone marrow-derived mesenchymal stem cells using Pluronic F-127 hydrogel in vitro,” Biomaterials, 2008, 29(5):573-579). The thermoreversible and promising drug release characteristics of poloxamer 407 render it an attractive candidate as a hydrogel scaffold for tissue engineering. However, because it is a completely synthetic and nonionic polymer, cells embedded in poloxamer 407 become unevenly distributed and clustered after several days of culture even when combined with collagen. (Id.) This characteristic can severely limit its potential use as a biomaterial in medical applications.
A class of biomaterials comprised of spontaneously self-assembling short (8-24 amino acids) ionic complementary oligopeptides has been described. (Zhang S, “Fabrication of novel biomaterials through molecular self-assembly,” Nat Biotechnol, 2003, 21(10):1171-1178; which is hereby incorporated by reference in its entirety, including all description on the peptides.) Self-assembling peptides form stable β-sheet structures when dissolved in deionized water. Exposure to electrolyte solutions initiates β-sheet assembly into interweaving nanofibers, producing a hydrogel containing up to >99% water content. (Zhang S et al., “Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane,” Proc Natl Acad Sci USA, 1993, 90(8): 3334-3338; Zhang S et al., “Self-complementary oligopeptide matrices support mammalian cell attachment. Biomaterials,” 1995, 16(18):1385-1393; Holmes T C et al., “Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds,” Proc Natl Acad Sci USA, 2000, 97(12):6728-6733; which are hereby incorporated by reference in their entirety, including any disclosure on peptides and methods for forming hydrogels). The structure of such nanofibers is about 3 orders of magnitude smaller than most polymer microfibers. (Kisiday J et al., “Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: implications for cartilage tissue repair,” Proc Natl Acad Sci USA, 2002, 99(15):9996-10001.) This important feature helps support cell attachment and differentiation of a variety of mammalian primary and transformed cells, such as neurons, chondrocytes, and microvascular endothelial cells. (Zhang et al., (1995); Kisiday et al., (2002); Semino C E et al., “Functional differentiation of hepatocyte-like spheroid structures from putative liver progenitor cells in three-dimensional peptide scaffolds,” Differentiation, 2003, 71(4-5):262-270; Semino C E et al., “Entrapment of migrating hippocampal neural cells in three-dimensional peptide nanofiber scaffold,” Tissue Eng, 2004, 10(3-4):643-655.)
This class of biomaterial has several advantages when used as a scaffold for tissue engineering. First, such a nanofiber network resembles ECM and provides a truly 3-D environment for cells to grow, migrate, proliferate and differentiate. Second, biomolecules in such a nanoscale environment diffuse slowly and are likely to establish a local molecular gradient more closely mimicking the in vivo scenario. Third, the degradation products of such peptide scaffolds are naturally occurring amino acids, potentially reducing their cytotoxicity. (Holmes et al. (2000).) In addition, the mechanical strength as well as chemical composition of the scaffold can be controlled through manipulation of peptide parameters. (Holmes et al. (2000); Leon E J et al., “Mechanical properties of a self-assembling oligopeptide matrix,” J Biomater Sci Polym Ed, 1998, 9(3):297-312; Caplan M R et al., “Effects of systematic variation of amino acid sequence on the mechanical properties of a self-assembling, oligopeptide biomaterial,” J Biomater Sci Polym Ed, 2002, 13(3):225-236; which are hereby incorporated by reference including disclosure relating to the relationship between peptide composition and length with mechanical strength or definition of structure.)
In terms of amino acid length, shorter peptides offer the advantage of lower cost, greater ease of synthesis, and higher solubility and purity. Furthermore, shorter peptides show less structural and chemical complexity, which facilitates their study. On the other hand, shorter peptides are less stable than longer ones, do not form well-ordered structures, and show fewer tendencies for self-assembly. They demonstrate variable solubility in water and sometimes precipitate into disordered aggregates. Researchers have concluded that designing shorter self-assembling peptides with well-defined structures represents a serious challenge (Leon et al., 1998).
EFK8 is one of the smallest peptides in this new family of biomaterials originally discovered by Zhang et al. (1993). EFK8 has an amino acid sequence that alternates between hydrophobic side chains and charged side chains, forming a special left-hand double helix that spontaneously undergoes association under physiological conditions (Zhang et al., “Design of nanostructured biological materials through self-assembly of peptides and proteins,” Curr Opin Chem Biol 2002; 6:865-71.).